CRISPR/Cas9 knockout of N-acetyltransferase 1 influences mesenchymal transition in glioblastoma in a cell line dependent manner

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and carries a poor prognosis despite standard of care treatment with surgical debulking, chemotherapy, and radiotherapy. Proneural to mesenchymal transition (PMT) is a key driver of GBM invasion and treatment resistance, yet its underlying molecular mechanisms remain uncharacterized. N-acetyltransferase 1 (NAT1) is implicated in the progression of several cancers, but its role in GBM is unknown. This study aimed to determine whether NAT1 regulated PMT phenotypic and transcriptional changes in GBM. NAT1 knockout (KO) cells were generated using CRISPR/Cas9 lentiviral delivery in three GBM cell lines (U87, U118, and A172). Cell proliferation was measured via manual cell counts and PrestoBlue assays measured cell viability. Morphological changes were assessed via microscopy, and GFAP and nestin immunofluorescence. PMT associated gene expression was profiled using RT-qPCR arrays. TGF-β1-treated control cells served as a positive control for PMT induction. NAT1 KO cells displayed altered morphology, characterized by decreased cluster formation. RT-qPCR array profiling revealed mRNA expression consistent with upregulation of IL1RN and MMP3 across U87 and U118 KO clones. However, overall gene expression profiles qualitatively suggested a mixed PMT phenotype for all three cell lines. NAT1 KO mediated induction of IL1RN and MMP3 indicates a conserved inflammatory and extracellular matrix axis that may broadly contribute to PMT in GBM. This raises important clinical considerations given glioma’s limited treatment options. Consequently, therapeutically inhibiting NAT1 emerges as a promising strategy to target GBM PMT, with broader implications for other cancers.